HPLC avidin monomer affinity resin

ABSTRACT

Novel, improved ligand-containing media, a method of preparation and use in the production of peptides, proteins, and the like, by chromatographic separation, and more specifically media having permanently attached via a covalent bond to an inert solid substrate an avidin polypeptide ligand in the dissociated renatured form which reversibly binds to certain molecules such as proteins, peptide, nucleotides, oligonucleotides, and the like and to other molecules which bind to avidin via biotinylation or by way of their secondary/tertiary micromolecular structures.

This application is a continuation, of application Ser. No. 414,785,filed Sep. 29, 1989, now abandoned.

BACKGROUND OF INVENTION

The present invention relates to improved ligand-containing media, theirmethod of preparation and use in the production of peptides, proteins,and the like, by chromatographic separation. In a preferred embodimentthe invention comprises media having permanently attached via a covalentbond to an inert solid substrate an avidin polypeptide ligand in thedissociated renatured form which reversibly binds to certain molecules(proteins, peptides, nucleotides, oligonucleotides, and the like) andother molecules which bind to avidin via biotinylation or by way oftheir secondary/tertiary micromolecular structures.

The production of certain peptides and proteins for use in human health,animal health, industrial, food, and agricultural markets has beenhampered by high cost and scarcity, particularly in the health carearea. Peptides and proteins for human health uses are naturallysynthesized by living organisms for their own needs and until recently,animals, plants, cadavers, serum and urine were the only sources fromwhich these valuable biomolecules could be obtained. For example,porcine or bovine insulin was extracted from the pancreas of pigs orcattle for use by diabetics and HGH (human growth hormone) was obtainedin small quantities from cadavers to treat infantile dwarfism. Thesebiomolecules were usually obtained in very small quantities because onlylimited amounts were produced biologically or they were rapidly degradedby enzymes in their environment. Two new technologies have beendeveloped which make possible the production of almost any peptide orprotein in relatively large quantities: chemical and biologicalsynthesis.

The chemical route is achieved by solid phase (Merrifield technique) andsolution phase peptide synthesis; this approach is usually limited topeptides of less than 20 amino acid residues. Biological synthesis usesgenetic engineering and recombinant DNA technologies and production ofcells in tissue cultures or by microbial fermentation. The biologicalroute has been the only practical approach to the production of highermolecular weight peptides in relatively large quantities.

Since interest in these biomolecules is based on their performance,purification of the desired biomolecule becomes a very important factor,especially in the health care and food additive industries, where thecost of purification alone, usually involving multiple process steps,can represent more than half of the total cost of producing the desiredbiomolecules.

The prior art has taught many techniques for covalently bindingmaterials such as proteins to solid substrates as a technique forseparating the bound species. For example, U.S. Pat. No. 4,732,811(granted Mar. 22, 1988) describes the use of polymers containingpolyaldehyde groups as capable of binding compounds containing primaryamino groups (e.g., protein, antibodies and drugs).

Regardless of the synthetic route used, purification is necessary andliquid chromatography has been the universal tool used for thesebioseparations. Among the chromatographic approaches available (ionexchange, size exclusion, reverse phase, hydrophobic interaction, andaffinity), affinity chromatography has the potential for significantlyreducing the number of purification steps required. Affinity columnsbased on avidin are known as being useful for the isolation of variousbiomolecules (D. A. Fuccillo, Biotechniques, 3 (6), 494-501 (1985)). Inparticular, avidin-biotin interactions have been applied to theisolation of proteins from biological synthetic routes. Avidin is abasic high-molecular weight glycoprotein found in egg whites; biotin isa low-molecular weight molecule with a fused imidazole-thiophene ringsystem which acts as a tag for recognition by avidin, resulting in anextremely stable avidin-biotin complex. Since the synthetic route chosenusually requires isolation of the desired biomolecule from very lowconcentrations in its environment, the extremely high affinity of avidinfor biotin has been exploited in the chromatographic concentration andisolation of biotin-tagged ("biotinylated") molecules by use of avidinaffinity columns. The specificity and affinity of avidin (nativetetrameric form of 4 identical subunits) for biotin is extremely highand chromatography columns based on this principle have been used foranalytical purposes. However, proteins and peptides containing thebiotin-tag can not be recovered from an avidin tetramer column withoutusing harsh conditions which invariably destroy the very biomoleculebeing isolated. Attempts to overcome the strong binding of biotin byavidin without losing the high specificity for binding biotinylatedmolecules have concentrated on using solid supports to stabilize thedissociated form of avidin; however, these columns have not beensatisfactory for preparative use because of the presence of severalclasses of binding sites with less than desirable binding capacities aswell as other deficiencies associated with the particular solid supportmatrices used (KP Henrikson, et al., Analytical Biochemistry, 94,366-370(1979)).

Various routes have been used to anchor the avidin moiety to a solidsupport. Most commonly, agarose activated to a proper form for covalentcoupling of the primary amino groups of avidin is used as a support (A.D. Landman, et al., J. Chem. Educ., 53 (9), 591 (1976)). However, thesecovalent linkages have not proven satisfactory due to chemicalinstability and resultant leaching of the avidin from the support, thusreducing the operational life of the column and also contaminating thepurified product sought. Other major disadvantages of these particularcolumns include nonspecific adsorption of proteins, compressibility ofthe column matrix at high liquid flow rates resulting in back pressureand reduced flow, and the sensitivity of agarose to microbialdegradation. In addition, agarose materials are not susceptible to easycleaning and sterilization. Other supports based on polystyrene orsilica (Japanese Kokai Patent Application JP 64-003129 A) have beenused, but these suffer from even lower binding capacities than doesagarose as well as incompatibility with certain biologically importantions.

For these reasons there is a need for a purification medium which allowsefficient separation and subsequent isolation of biologically importantmolecules in a form satisfactory for the critical needs of such fieldsas health care and food additives.

The terms "adsorption" or "chromatographic adsorption" are intended inthe specification and the appended claims in a broad, but perhaps notentirely pure technical, sense to embrace any form of binding betweenchemical species, other than covalent binding. Thus, binding by affinityor Van der Walls forces, while technically distinguishable, are bothintended to be included within the broad meaning of "adsorption" as usedherein.

SUMMARY OF INVENTION

It is an object of this invention to provide a method of isolation ofsynthetic or natural molecules, and/or biotinylated derivatives thereof,by adsorption of said molecules onto novel affinity media containingavidin fixed to a solid inert support. It is a further object to usecompositions for the affinity media which are based on chemicallystable, non-hydrolyzable linkages of avidin to a polymeric substrate.These objects, and others which will become apparent from the followingdisclosure, are achieved by the present invention which is, in oneaspect, a media remarkably useful for isolating biotinylated molecules,comprising an inert, crosslinked polymeric substrate having covalentlybonded thereto through a non-hydrolyzable linkage of the formula --NH₂CH₂ --, an avidin polypeptide ligand in the dissociated renaturedmonomeric form. In another aspect, the invention provides an improvedprocess for the preparation of avidin affinity media which comprisesreacting an excess of avidin with a polymeric substrate containingreactive formyl functional groups, chemically reducing the imino bondsto stable amine linkages, and chemically denaturing and renaturing thebound avidin to its monomeric form. A still further aspect of theinvention is an improved, process for separating synthetic or naturalmolecules from a fluid mixture, such as fermentation broth or reactionmixture, containing various by-products and impurities as well as themolecules, by means of adsorption elution using the novel media of thisinvention.

As contained herein and in the appended claims, the term "column" isused in the broad sense to define a container which holds adsorptionmedia. Typically, in chromatographic separations, the columns are madeof glass, silica, stainless steel or the like and are in the form ofhollow tubes (or capillary fibers) often wound in a spiral, or longcylindrical tubes having, at opposition ends, inlet and outlet means. Inpreparative chromatography or industrial separations the column may be avertical vessel, usually cylindrical, for housing a stationary bed ofadsorbent. The present invention is suitable for use with any particularcolumn configuration.

Prior art avidin-tetramer columns containing bound enzymes/proteins havelimited lifetimes due to loss of activity ("fouled" resin) at whichpoint the entire resin must be discarded due to the irreversible bindingof the enzyme in question. However, when avidin-monomer affinity columnsof the present invention are used to immobilize enzymes, the column canbe regenerated easily upon any loss of enzyme activity because of thereversibility of the avidin-monomer/enzyme complex.

DETAILED DESCRIPTION OF INVENTION

We have discovered a novel affinity chromatography composition, animproved process for preparation of the chromatographic media, and newuses of the novel media which allow unexpected and surprisingimprovements in the isolation and purity of natural and syntheticbiomolecules.

Preparation of the avidin monomer affinity media by the presentinvention is based on the attachment of avidin to a compatiblechemically inert substrate through a chemically stable linkage whichwill not degrade or dissociate during subsequent chemical treatmentswhich are necessary to release bound biomolecules. Suitable inert solidsubstrates for the improved media of the present invention include awide range of polymeric and inorganic solids. Preferably, the substratesshould be highly inert, porous and particulate (spherical preferred). Awell-known and highly preferred type of substrate is the porouscrosslinked organic polymeric adsorbent or ion exchange resin having aprecipitated or macroreticular structure. This type of particle iscommonly used in chromatographic separations as well as industrialpurification techniques. Illustrative materials are the Toyopearl resins(TM of TOSOH, Japan) and Amberlite XAD series of polymers (TM of Rohmand Haas Co., USA). Acrylic and styrene-based polymers and copolymers ofvery high porosity and surface area are a most preferred class ofsubstrates (see, for example, U.S. Pat. No. 4,382,124). Of the lesspreferred substrates may be mentioned, glass beads, silica, gelpolymers, and the like.

Suitable linking groups used to covalently bind avidin to the solidsubstrates include: --CH₂ NH--, --CONH--, --NHC(O)NH--,--C(O)NHNHC(O)NH-- and --SO₂ NH-- wherein the --NH-- moiety on the rightside of the aforesaid groups is contributed by the avidin. A highlypreferred type of linkage involves the reduced imino group of theformula --NHCH₂ --. Linking groups may also include alkylenescycloalkyl, aryl, aralkylene, or carboxyl, hydroxy or alkoxy substitutedderivatives thereof as spacer structures between the backbone of thepolymeric substrate and actual linking site of the avidin molecule.Representative structures would include: --NH(CH₂)n, --NH(CH₂)nC₆ H₄ --,where n=1 to 4, preferably where n=1 or 2. Linkage may involve anynumber of the amino groups found in the avidin polypeptide, e.g., theE-amino group of lysine, the imidazole group of histidine, or any of theα-amino groups of the N-terminal aminoacids. Equations I and IIillustrate the chemical reactions which take place to form the linkagebetween avidin and the polymeric substrate (in this case, containingformyl groups). ##STR1## The amino groups of avidin chemically reactwith appropriate functional groups positioned on the aforementionedpolymeric substrate, e.g., formyl groups, to produce the intermediateimino linkage, --CH═N--, which is subsequently treated with anappropriate reducing agent to produce the chemically stable aminelinkage of the formula --CH₂ NH--. Suitable reducing agents typicallyinclude methyl hydride complexes, NaCNBH₃, NaBH₄, H₂, and BH₃.Preferably, salts of cyanoborohydride are used to perform the reductionto the amino linkage.

An important characteristic of the avidin affinity medium is the natureof the avidin units bound to the inert support. Avidin occurs naturallyin a tetrameric form with four identical subunits, each consisting of128 aminoacid residues, six mannose residues, and three glucosamineresidues, for a combined molecular weight of approximately 68,000.Tetrameric avidin, even when bound to a polymeric substrate, such asagarose (a polysaccharide), forms extremely stable complexes withbiotinylated molecules (dissociation constant, K(d), of 10(-15)),rendering bound biotinylated enzymes, peptides, and the like, almostimpossible to recover in high yields and purity due to the chemicallyaggressive reactions which must be used to release the boundbiotinylated molecules from their complexes with the tetrameric avidinmedia. It is, therefore, a characteristic of the present invention thata predominant amount of the bound avidin be present in its monomericform, in which the dissociation constant for the avidin-biotin complexis considerably greater than 10(-15), preferably greater than 10(-10),and most preferably, between 10(-9) and 10(-7). It is believed that theunique combination of the chemically stable linkage of the avidin to thepolymeric substrate, chemical structure of the substrate (hydrophobicinteractions, hydrogen bonding, and the like), and the physicalstructure of the substrate (porosity, crosslinking level, and the like)are responsible for establishing the spatial constraints on a molecularlevel which maintain the bound avidin in its monomeric form where itshigh specificity for the biotin group is maintained, while the greaterdissociative characteristics of the monomeric avidin-biotin complex makeisolation and recovery of biotinylated molecules truly reversible.

The process for preparing the novel monomeric avidin affinity media ofthe invention is based on the introduction of the chemically stableamino linkage between the substrate and avidin, followed by well knowndenaturing and renaturing treatments which produce the finalbound-monomeric avidin affinity medium. The first stage of the processinvolves the reaction of avidin (normally an excess) with the functionalgroups of the polymeric substrate. The parameters which control theextent to which the avidin is bound to the substrate in this stepinclude time, pH, temperature, concentration of reactants (avidin,substrate functional groups, reducing agent), and composition ofsubstrate.

Reaction temperatures are limited due to the sensitivity of mostproteins to moderate temperatures; however, avidin is extremelytemperature insensitive and a range of 5 to 35 C. may be typically used;since most reactions proceed more slowly at reduced temperatures, 20-25C. is used to take advantage of the faster rates allowed by avidin'sgood temperature stability. Reaction times of less than 10 hrs result inlower levels of avidin fixation, while times in excess of 15 hrs do notappreciably increase the fixation of avidin. Reaction times of 24 hrsmay be used to ensure maximum avidin uptake without encounteringcounterproductive side reactions.

A pH range of 5 to 10 may be used to carry out the avidin fixationreaction. The range of 6.5 to 8 is most suitable for balancing thetendency of charged functional groups present in the protein to formintramolecular bonds and the need to maximize the concentration of freeunprotonated amino groups for reaction with the substrate formyl groups.A suitable buffer which maintains the pH in this region is preferable,with pH 6.5-7.5 being most preferable.

The concentration of avidin (mg/mL resin) charged to load the proteinonto the substrate containing formyl functional groups may range from0.5 to 10 mg/mL. Concentrations of 2 to 5 mg/mL are preferred in thatthe ratio of tetrameric avidin to amino linkages is maintained such thatsubsequent conversion to bound monomeric avidin is favored. Theconcentration of formyl groups in the substrate polymer may vary over awide range: 10 to 100 μmoles --CHO/mL resin; concentrations of 35-70μmoles/mL are preferred in that a good balance is established regardingthe number of amino linkages created between the polymeric substrate andthe bound avidin while maintaining the spatial constraints within theavidin-polymer matrix which allow ready conversion to the monomeric formof the bound avidin. The concentration of reducing agent may vary froman equimolar to a tenfold molar excess in relation to the formyl groupconcentration. Molar ratios (reducing agent/formyl group) in excess of 5result in less of the bound avidin being converted to its monomericform; ratios from 1-3, corresponding to about 3-10 mg reducing agent/mL(when using sodium cyanoborohydride at formyl group concentrations of 55moles/mL, for example), represent a preferred range with regard toconverting the bound avidin to its monomeric form.

Conversion of the bound avidin from its tetrameric form to the monomericform is accomplished by conventional denaturing/renaturing treatmentsusing a variety of reagents: such as aqueous DMSO, urea, lithiumchloride, guanidine HCl. Preferably, a solution of guanidinehydrochloride, containing about 10% vol/vol acetic acid, pH 2, may beused to convert the bound avidin into its monomeric form.

Isolation and purification of natural and synthetic molecules using theavidin affinity media may be achieved by contacting aqueous or organicmixtures containing the desired target molecule together with otherundesirable constituents with particles containing the composition ofthe present invention, i.e., bound monomeric avidin. Proteins, peptides,enzymes, nucleotides, oligonucleotides, and their correspondingbiotinylated recombinant versions may be isolated and purified, andquantitated for analytical purposes using avidin affinitychromatography. In addition the novel affinity media may be used for:the localization and separation of antigens, development of immunoassaytechniques, and production purification and/or recovery of DNA or RNA orpurified probe molecules for hybridization studies, and purification orsequencing of genetic information from a variety of organisms, or otherapplications which would benefit from the use of avidin affinitychromatography.

The avidin affinity media may be used in several forms to achieve theaforementioned separations. Most commonly, media would be used in theform of particulate beads, ranging in size from 5 microns up to about1000 microns. In addition, the media may be used either in a columnoperation or a batch mode. For example, in the batch mode, fermentationbroths of recombinant proteins may be treated with a quantity of theavidin affinity media to remove secreted proteins of interest; thetreated fermentation broth may be recombined with other streams to betreated (recycled) to remove as much of the target molecules aspossible. Batch mode operation is particularly useful for thepreparative scale isolation of relatively large quantities ofbiomolecules. On the other hand, the column mode of operation ispreferably used for analytical and preliminary evaluation purposes aswell as small scale preparative needs.

Once the target molecule has been isolated, i.e., concentrated onto theavidin affinity medium, it must be removed from the media and separated.This step is carried out by any number of conventional processes, whichare well known to those familiar with bioseparations. Among theprocesses used to elute target molecules from the affinity media aretreatment with solutions of urea, glycine, acetic acid, varying saltcontent, biotin, varying pH, and the like.

Target molecules which may be isolated and purified by use of the avidinaffinity media possess one of several characteristics which allow theseparation to take place. One characteristic is the presence of thebiotin group in the molecule to be isolated. D(+)Biotin (Structure I),also known as Vitamin H or coenzyme R, has a molecular weight of 244 andchemically reacts (by known reactions) through its carboxyl group withthe amino groups of enzymes, peptides, proteins, and the like, to anchoritself to the target molecule through the resultant amide linkage(Equation II). Other forms of Biotin e.g., imino biotin or lipoic acidcan also aid in the separation of biomolecules. ##STR2##

The resultant molecule of Equation II is now biotinylated, i.e., itcontains the biotin group. The biotin group acts as an identifier whenthis molecule is then exposed to the avidin monomer affinity medium; thestrong complexation of avidin with biotin causes the biotinylatedmolecule to be absorbed onto the medium and thus separated from allother non-biotinylated molecules present in the particular mixture.Conventional methods (discussed previously) are then used to remove thebiotinylated molecule.

In order to more fully illustrate the nature of this invention and themanner of practicing the same, the following examples are presented.These examples represent just a few of the many uses and compositions ofthe invention; they are intended to be illustrative but not limiting.Various modifications, alternatives, and improvements should becomeapparent to those skilled in the art without departing from the spiritand scope of the invention.

EXAMPLE 1 General Procedure for Preparing Avidin Affinity Columns

Acrylic resin (particle size: 44-88μ) containing 55 μmole formyl (--CHO)groups/mL resin, AF--formyl Toyopearl(TM) 650M (TOSOH) was placed on asintered glass filter and washed with approximately 10 bed volumes of100 mM potassium phosphate buffer solution (pH=7.5); the wet resin wastransferred to a polypropylene bottle. Purified avidin (Sigma ChemicalCo.) was weighed out and dissolved in 100 mM phosphate buffer solution(pH=7.5); final avidin concentration was measured by absorbance valuesat 282 nm (E(1%)=15.5). Avidin (4.0 mg/mL resin) was added to theprewashed resin in the polypropylene bottle, the mixture stirred gentlyfor 10-15 minutes, and sodium cyanoborohydride (7.5 mg/mL resin) wasthen added. The bottle was capped and placed in a horizontal shaker(slow speed) maintained at 23-25 C. for 24 hrs. The modified resin wasthen transferred to a column and then washed with 100 mM potassiumphosphate buffer at pH=7.5, the eluate recovered and measured forunbound avidin (absorbance method); the total amount of resin-boundavidin was then calculated by difference.

The avidin-loaded resin was then washed with 4M guanidine HCl solutioncontaining 10% acetic acid (vol/vol), pH 2, to dissociate the tetramericavidin into its monomeric form. Eluates were collected and the amount ofavidin removed from the resin was determined via absorbance readings.The avidin monomer affinity resin was then slurry packed into HPLCcolumns (Upchurch Scientific, Inc.) for subsequent use in thepurification of proteins.

EXAMPLE 2 Evaluation of Avidin Monomer Affinity Column Performance

Total biotin binding capacity was estimated by calculation of the amountof avidin monomer immobilized per ml of resin. Biotin binding affinitiesand capacities were determined using D-(¹⁴ C) biotin. Crude extracts of24 hr cultures of E. coli containing the plasmid ptac 1.3 t were used todetermine the binding capacity for biotinylated peptides (V. L. Murtif,et al, Proc. Natl. Acad. Sci., USA 82, 5617-5621 (1985)) and to evaluatethe column regarding purification of recombinant proteins.

Characteristics of resin evaluated:

Avidin monomer capacity (calc)=74.9 nmoles/mL

Total ¹⁴ C biotin binding capacity=58.7 nmoles/mL

Reversible ¹⁴ C binding capacity=51.2 nmoles/mL

Revesible Biotinylated protein (1.3S_(e)) cap=68.4 nmoles/mL

Approximately 78% of resin immobilized avidin binds biotin and 87% ofthese do so reversibly; with the 1.3S_(e) peptide, 91% of the avidinshowed reversible binding capability, indicating almost completeconversion of avidin to the monomeric form (reduced/reversible binding).

EXAMPLES 3-6 Various Conditions Used to Prepare the Avidin AffinityColumns

In a manner similar to that described in Example 1, different avidinaffinity columns were prepared under a variety of conditions andevaluated for their effectiveness in binding proteins (according toExample 2). Concentrations are expressed per mL of resin.

EXAMPLE 3

In a manner similar to that described in Example 1, an acetate bufferwas used (pH 5.5), avidin was added at 2.3 mg/mL, and sodiumcyanoborohydride was added at 7.5 mg/mL: Characteristics of resinobtained:

Avidin monomer capacity (calc)=57 nmoles/mL

Protein 1.3S₃ binding capacity=21.5 nmoles/mL

EXAMPLE 4

In a manner similar to that described in Example 1, a phosphate bufferwas used (pH 7.5), avidin was added at 4.0 mg/mL, and sodiumcyanoborohydride was added at 7.6 mg/mL:

Characteristics of resin obtained:

Avidin monomer capacity (calc)=75 nmoles/mL

Protein 1.3S_(e) binding capacity=64 nmoles/mL

EXAMPLE 5

In a manner similar to that described in Example 1, a phosphate bufferwas used (pH 6.5), avidin was added at 3.85 mg/mL, and sodiumcyanoborohydride was added at 23.0 mg/mL:

Characteristics of resin obtained:

Avidin monomer capacity (calc)=161 nmoles/mL

Protein 1.3S_(e) binding capacity=115 nmoles/mL

EXAMPLE 6

In a manner similar to that described in Example 1, a buffer oftris(hydroxymethyl)aminomethane was used (pH 7.8), avidin was added at3.0 mg/mL, and sodium cyanoborohydride was added at 30.0 mg/mL:

Characteristics of resin obtained:

Avidin monomer capacity (calc)=43 nmoles/mL

EXAMPLE 7 Preparation of Protein Samples/Purification by AffinityChromatography

A. Recombinant Biotinyl Subunit from E. Coli

A crude extract was prepared by passing a suspension of cells (10 g ofCSR26 E. coli which over express the 1.3S_(e) subunit) in Buffer A (100mM ammonium bicarbonate, 1.0 mM ethylenediamine tetraacetate disodiumsalt, 2.0 mM PMSF (phenylmethylsulfonyl fluoride), 0.01% sodium azide,and 1.0 mM DTT (dithiothreitol), pH 8.3) through a French Press or bylysis by sonication. This procedure was carried out twice and the celldebris removed by centrifugation. The clear supernatant was treated withstreptomycin sulfate to remove nucleic acids and then fractionated bydifferential ammonium sulfate saturation. The resulting protein pelletfrom the 30-60% ammonium sulfate saturation contained the biotinylated1.3S_(e) proteins and was dissolved in 14 mL of Buffer A. This solutionwas then dialyzed against Buffer B (100 mM potassium phosphate, 0.15Msodium chloride, pH 6.8).

B. Transcarboxylase Biotinyl Subunit from Propionibacterium Shermanii

A crude extract of transcarboxylase was prepared as described by H. G.Wood, B. Jacobson, B. I. Gerwin, and D. B. Northrup in Methods Enzymol.,13, 215-231 (1969).

EXAMPLE 8 Purification of Biotinylated 1.3S_(e) Subunit andTranscarboxylase from Crude Extracts A. General Method

High Peformance Liquid Chromatography (HPLC) was used to characterizethe quality of separation and recovery of the proteins and peptidesafter the crude extracts were subjected to affinity chromatography:Shimadzu HPLC system with a variable wavelength detector (monitored at220 nm). Other chromatographic methods were optionally employed tofurther characterize the purification of the crude extracts: reversephase HPLC (Synchropak RP-C4 column, 0.1% trifluoroacetic acid(TFA)/water and 0.1% TFA/acetonitrile solvent system); hydrophobicinteraction chromatography (HI-HPLC) using a Progel-TSK Ether 5PW(Supelco, Inc.) column with a two solvent system (2.0M ammonium sulfatein 100 mM potassium phosphate buffer (pH 6.8) and 100 mM potassiumphosphate buffer (pH 6.8)).

The binding capacities of various avidin affinity columns were evaluatedby equilibrating the column with Buffer B (described in Example 7) andsaturating the column by multiple injections of known concentrations ofcrude extracts (described in Example 7). The columns were next washedextensively with Buffer B until the absorbencies of the eluates at 220nm were reduced to 0.01 OD (optical density). The columns were thenwashed with Buffer C (100 mM glycine-HCl buffer, pH 2.0) to elutepreviously bound 1.3S_(e) biotinyl subunit. SDS-PAGE was used to verifyidentity of the subunit.

B. Affinity Chromatography Columns Evaluated

A column prepared according the present invention (Example 1),designated Avidin-HPLC, and one representing prior art technology,designated Avidin-Agarose (Sigma Chemical Co., subunit of avidinattached to 4% crosslinked agarose beads) were evaluated side by side.

C. Column Performance (recombinant 1.3S_(e) subunit from E. coli)

The biotin and protein contents of the fractions eluted with Buffer Cusing the affinity medium of the present invention (Avidin-HPLC) and aconventional affinity medium (Avidin-Agarose) were determined by theaforementioned methods. Bed volumes used were 1.26 and 5.0 mL for theAvidin-HPLC and Avidin-Agarose columns, respectively; flow rates usedwere 1.0 mL/min.

Operating conditions for the affinity columns included several importantparameters. Prewashing the columns prior to loading was typicallyrequired: equilibration with 4 bed volumes of 100 mM potassium phosphatebuffer (pH 6.8) containing 150 mM sodium chloride and biotin (1.0mg/mL), followed by elution with 10 bed volumes of Buffer C, theseconditions were also used to regenerate new or stored columns.

Loading the protein sample was considered complete when column washesgave absorbance values of less than 0.05 OD for the Avidin-Agarosecolumn and 0.01 OD for the Avidin-HPLC column. Elution of the boundproteins was accomplished with Buffer C and absorbance readings wereagain used to determine endpoints for the elution process. Repeated useof the same column resulted in significant differences between the twotypes of columns: column shrinkage for the Avidin-Agarose system and asteady decrease in binding capacity over 6 cycles whereas no shrinkagewas observed for the Avidin-HPLC column over 10-15 cycles with noreduction in binding capacity.

The binding capacity of the Avidin-HPLC column was 4 times greater thanthat of the conventional Avidin-Agarose column and could be operated 6times faster (40 min versus 4 hrs per cycle, after prewash). Nodegradation of the Avidin-HPLC column's capacity was observed over thelifetime of these studies whereas the conventional resin was less than50% of initial capacity after 6 cycles. A summary of the results usingthe recombinant biotinyl subunit from E. coli can be found in Table 8C.

                  TABLE 8C                                                        ______________________________________                                        Column:        Avidin-HPLC  Avidin-Agarose                                    ______________________________________                                        Total protein capacity                                                                       42           11                                                (1.3S subunit, nmoles/mL)                                                     Fraction of capacity as                                                                         0.28         0.28                                           biotinylated 1.3S.sub.e subunit                                               Operation Times (min):                                                        Prewash        25           125                                               Load/wash      15           120                                               Elution        15           60                                                Regeneration   10           60                                                Relative binding capacity                                                                      1.0           0.42                                           after six regeneration                                                        cycles (1.0 = no change):                                                     Storage Stability                                                                            Stable       Unstable**                                        (Water, 25° C.):                                                       ______________________________________                                         **recommended storage in 10 mM sodium phosphate buffer (pH 6.8), 50%          glycerol, 150 mM sodium chloride and 0.02% sodium azide at -20° C.

D. Column Performance (isolation of transcarboxylase containing the1.3S_(e) subunit from P. shermanii)

Purification of the biotinyl enzyme, trans-carboxylase from extracts of(P. shermanii), was attempted in the same manner as that of therecombinant 1.3S_(e) subunit from E. coli. However, in this case none ofthe enzyme was recovered by using the conventional known resin(Avidin-Agarose) while 25 to 50% pure enzyme was achieved by using theAvidin-HPLC resin. Table 8D summarizes performance characteristics ofthe two resins with regard to P. shermanii transcarboxylase enzymepurification.

                  TABLE 8D                                                        ______________________________________                                        Column:          Avidin-HPLC Avidin-Agarose                                   ______________________________________                                        Total protein capacity                                                                         42          --                                               (1.3S.sub.e subunit, nmoles/mL)                                               Specific activity of recovered                                                                 8-16**      0                                                enzyme (μmoles/min/mg                                                      protein)                                                                      ______________________________________                                         **30 μmoles/min/mg protein is equivalent to 100% pure transcarboxylase

E. Discussion of Results

Table 8C summarizes the capacity (1.3S_(e) subunit from E. coli) andoperation time advantages of the avidin affinity medium of the presentinvention.

Table 8D summarizes the enrichment in purity achieved during theisolation of transcarboxylase enzyme (P. shermanii) using the avidinaffinity medium of the present invention. In contrast, the conventionalAvidin-Agarose medium does not provide any enrichment at all.

The following specific examples illustrate variations in the synthesesof the novel ligand-containing media of the invention. In particular,acrylic backbone polymers containing formyl (--CHO) groups can be madeby the technique of A. Kanamori et al., described in J-Chromatography,363 231-242 (1986); this procedure was used in Examples 9 and 10 whichfollow:

EXAMPLE 9 Preparation of Formyl Group (CHO) Containing Substrate Basedon Acrylic Backbone Polymer

AF-epoxy Toyopearl (TM) 650M resin (dry, 45-90 microns, 10.0 g)containing 89 μmoles/g of epoxy groups, was added to a mixture of 5.0 gof dextrose (glucose) and 40 mL of 0.1M sodium hydroxide in a 4-oz jar.The closed container was then incubated at 40C for 24 hrs in aheater/shaker apparatus (200 rpm). The resultant resin was placed in acolumn, washed thoroughly with water and transferred to a 4-oz jar.Sodium periodate solution (0.1M, 15 mL) was then added and the resultantmixture shaken in an ice bath for 1 hr. The beads were washed with wateron a Buchner funnel and then incubated in 25 ml of 0.1M HCl at 25 C. for30 min in a heater/shaker apparatus. The beads were finally washedthoroughly with water; formyl group analysis indicated a --CHO contentof 55 μmoles/g (dry resin).

EXAMPLE 10 Preparation of Formyl Group (CHO) Containing Substrate Basedon Acrylic Backbone Polymer

In a manner similar to Example 9, 10.0 g of AF-epoxy Toyopearl™ 650Mresin was mixed with 100 g of 0.1M sodium hydroxide solution containing0.020 g sodium borohydride in an 8-oz jar. The mixture was incubated at40 C. in a heater/shaker apparatus (200 rpm) for 24 hrs. The resultantresin was placed in a column, washed thoroughly with water andtransferred to a 4-oz jar. Sodium periodate solution (0.1M, 15 mL) wasthen added and the resultant mixture shaken in an ice bath for 1 hr. Thebeads were washed with water on a Buchner funnel and then incubated in25 mL of 0.1M HCl at 25 C. for 30 min in a heater/shaker apparatus. Thebeads were finally washed thoroughly with water; formyl group analysisindicated a --CHO content of 55 μmoles/g (dry resin).

EXAMPLE 11 Preparation of Formyl Group (CHO) Containing Substrate basedon Styrenic Backbone Polymer A. Copolymer Composition

A macroporous copolymer containing chloromethyl groups was prepared bysuspension polymerization of 55% vinylbenzyl chloride (VBC), 36%divinylbenzene (DVB), 9% ethylvinylbenzene (EVB); pentanol (40% vol) andtoluene (20% vol) were used as porogens (phase extender). The productcopolymer contained 8.2% Cl.

B. Conversion to Formyl Group Containing Polymer

Using a procedure described by J. T. Ayres and C. K. Mann in Journal ofPolymer Science, Polymer Letters, 3, 505-508 (1965), the chloromethylgroups of the styrenic copolymer were converted to formyl (--CHO) groupsby dimethylsulfoxide (DMSO) oxidation. Ten grams of the chloromethylatedresin (described above) was mixed with 14 grams of sodium bicarbonate in200 mL of DMSO at 155 C. for 6 hrs. The product was filtered, washedwith DMSO, hot water, and acetone, and dried at 100 C. under vacuum.

EXAMPLE 12 Preparation of Formyl Group Containing Substrate Based onStyrenic Backbone Polymer A. Copolymer Composition

In a manner similar to Example 11, a macroporous copolymer of thefollowing composition was prepared: 29 VBC/38 DVB/9 EVB/24 Styrene (S)with porogen levels the same as Example 11. The product copolymercontained 5.4% Cl.

B. Conversion to Formyl Group Containing Polymer

In a manner similar to Example 11, the above chloromethylated copolymerwas converted to the formyl derivative.

EXAMPLE 13 Preparation of Formyl Group (CHO) Containing Substrate Basedon Styrenic Backbone Polymer A. Copolymer Composition

In a manner similar to Example 11, a macroporous copolymer of thefollowing composition was prepared: 15 VBC/39 DVB/10 EVB/36 S withporogen levels the same as Example 11. The product copolymer contained2.7% Cl.

B. Conversion to Formyl Group Containing Polymer

In a manner similar to Example 11, the above chloromethylated copolymerwas converted to the formyl derivative.

EXAMPLE 14 Preparation of Formyl Group (CHO) Containing Substrate Basedon Styrenic Backbone Polymer A. Copolymer Composition

In a manner similar to Example 11, a macroporous copolymer of thefollowing composition was prepared: 7 VBC/40 DVB/10 EVB/43 S withporogen levels the same as Example 11. The product copolymer contained1.6% Cl.

B. Conversion to Formyl Group Containing Polymer

In a manner similar to Example 11, the above chloromethylated copolymerwas converted to the formyl derivative.

I claim:
 1. A novel ligand-containing medium for chromatographicadsorption comprising a crosslinked organic polymer having covalentlybonded thereto through a chemically stable, non-hydrolyzable linkinggroup an avidin polypeptide ligand in the dissociated renatured formwherein the avidin ligand is predominantly in a monomeric form whichbinds with biotin to form a complex with a dissociation constant of notless than about 10⁻¹⁰ Molar and wherein the crosslinked organic polymeris capable of maintaining the avidin ligand in the monomeric form. 2.The medium of claim 1 wherein the non-hydrolyzable linking group is acarbon-nitrogen group or a sulfur-nitrogen group.
 3. The medium of claim1 wherein the non-hydrolyzable linking group is selected from the groupconsisting of --CH₂ NH--, --CONH--, --NHC(O)NH--, --C(O)NHNHC(O)NH-- andSO₂ NH.
 4. The medium of claim 3 wherein the avidin ligands are attachedthrough the linking groups to a crosslinked organic polymer.
 5. Themedium of claim 1 wherein the crosslinked organic polymer is a porousadsorbent or ion exchange resin in particulate form.
 6. The medium ofclaim 5 wherein the crosslinked organic polymeric adsorbent or ionexchange resin in particulate form is derived primarily from an acrylicor styrenic monomer.
 7. The medium of claim 1 wherein the avidinaffinity group is in a monomer form which binds with biotin to produce acomplex with a dissociation constant of not less than 10⁻⁹ molar.
 8. Anaffinity chromatographic column which comprises a tubular container withinlet and outlet means at opposite ends of the tube and having fixedwithin the tube the novel ligand-containing medium of claim
 1. 9. Amethod for isolating synthetic or natural molecules from a fluid mixturecontaining the same wherein the molecules have an affinity for avidin,or may be biotinylated to have an affinity for avidin or theirrecombinant versions having affinity for avidin or biotinylatedrecombinant versions which comprises passing the fluid mixture intointimate contact with the ligand containing medium of claim 1 andsubsequently eluting the adsorbed synthetic or natural molecules fromthe ligand containing medium.
 10. The method of claim 9 wherein thefluid mixture is an organic or aqueous liquid mixture.
 11. The method ofclaim 9 wherein the synthetic or natural molecules consist of peptides,proteins, nucleotides, oligonucleotides, or recombinant or biotinylatedor recombinant-biotinylated versions thereof.
 12. The method of claim 9where the fluid mixture containing the synthetic or natural molecules iscontacted with the ligand-containing medium of claim 1 contained withina chromatographic column.
 13. The method of claim 9 wherein a liquidmixture of synthetic or natural molecules is separated by liquidaffinity chromatography in a column comprising the medium of claim 1.